ARTICLE TOOLS

Abstract

Deoxyribonucleic acid (DNA) cloning is the art of creating recombinant DNA molecules that can be introduced into living cells, replicated and stably inherited, such that multiple ‘clonal’ copies of that DNA are produced. Here, DNA of interest is spliced into a DNA vector that has the ability to replicate in host cells and be inherited into future generations of those cells.

This article describes the theory behind cloning; the preparation of vector and insert DNA for cloning; the techniques that can be used to create recombinant DNA molecules (including traditional ligation of blunt and sticky ends, TA cloning, topo cloning, ligation-independent cloning, recombineering and gateway cloning); introduction of recombinant DNA molecules into host cells; the major classes of vectors available and the variety and utility of the various features and that can be present in those vectors.

Key Concepts:

DNA can be joined, or ligated, together to form a recombinant DNA molecule.

When cloning DNA, that DNA is ligated to a vector molecule allowing it to be subsequently immortalised.

Recombinant vector:DNA constructs can be introduced into living cells, so-called host cells, through the process of transformation.

Once introduced into host cells, the DNA and vector construct is immortalised and can be replicated and stored.

Different vectors allow for introduction and maintenance in different host cells.

A range of vectors are available, or can be constructed, that contain a variety of elements that confer upon that clone its usefulness. Such elements may include one or more of the following: sequences that control replication and inheritance within the host cell and sequences that allow selection of the recombinant molecule, for example, antibiotic resistance genes, reporter genes and promoter sequences that drive expression of a gene that has been cloned.